This invention relates to the microbial degradation of halogenated aromatic compounds, and more particularly to a simple assay method for detecting such degradation.
Halogenated aromatic compounds are a genus of organic compounds including, for example, halogenated dioxins, halogenated dibenzofurans and halogenated biphenyls. Such compounds, or related molecular species containing such compounds as impurities, have been used in various ways in industry, but there are concerns about their possible biological effects on humans and other species.
A subgenus of such compounds is the mono- and polychlorinated biphenyls, sometimes generically designated "polychlorinated biphenyls" or "PCBs", which were in wide use as dielectric materials in capacitors and the like. After such use ceased, sizeable quantities of these compounds were discarded in landfills and waterways. Many of them are not rapidly biodegraded and further concern has been expressed about possible long-term effects of the discarded materials on the environment.
A common microbial degradation route for halogenated aromatic compounds, the 2,3-dioxygenase pathway, is effective with many mono-, di- and trihalogenated compounds. As applied to PCBs, it includes the following steps performed under aerobic conditions: (1) dioxygenase enzyme activity to insert two oxygen atoms at the 2- and 3-positions, forming a 2,3-dihydrodiol; (2) dihydrodiol dehydrogenase activity to re-aromatize the 2,3dihydrodiol to a 2,3-dihydroxybiphenyl; (3) meta-cleavage of said 2,3-dihydroxybiphenyl by 2,3-dihydroxybiphenyl dioxygenase to produce a substituted hexadienoic acid such as 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, such compounds being brightly yellow in color by reason of their high degree of conjugation. In a fourth step characteristic of many microorganisms, a hydrase enzyme cleaves said hexadienoic acid to produce colorless products including a benzoic acid.
While the 2,3-dioxygenase pathway is generally effective for degradation of compounds relatively low in halogen, it is not effective for some compounds, including 2,5,2',5'-tetrachlorobiphenyl and 4,4'-dichlorobiphenyl. Such compounds can, however, often be dehalogenated to products which are degraded by the 2,3-dioxygenase pathway. Dehalogenation is typically performed by anaerobic microbes, in river or lake sediment or the like where little or no oxygen is present. Other methods, including chemical methods, for the dehalogenation of such compounds are also available. It is then possible to expose the dehalogenation products to oxygen and to the action of 2,3-dioxygenase active microbes for further degradation. Hereinafter, the dehalogenation of highly halogenated species under anaerobic conditions is designated "anaerobic dehalogenation". Further degradation via the 2,3-dioxygenase pathway is designated "dioxygenase degradation" and molecular species capable of being so degraded are designated "dioxygenase-susceptible" species, while species incapable of such degradation are designated "non-dioxygenase-susceptible" species.
As examples of anaerobic dehalogenation mechanisms, various microorganisms in soils and waterway sediments are capable of dechlorinating 2,5,2',5'-tetrachlorobiphenyl to such compounds as 2,5,2'-trichlorobiphenyl, 2,2'-dichlorobiphenyl and 2-chlorobiphenyl, all of which are dioxygenase-susceptible. Similarly, anaerobic dechlorination of 4,4'-dichiorobiphenyl to the dioxygenase-susceptible 4-chlorobiphenyl is possible.
It has typically been very difficult to determine, at least for qualitative purposes, whether dehalogenation of PCBs has taken place and the extent thereof. Such operations as solvent extraction, centrifugation and gas chromatographic analysis of the products have been necessary. These procedures are time consuming and are poorly suited for screening large numbers of samples.
Thus, there is a need for a simple screening method to determine whether or not a given material contains microorganisms capable of anaerobically dechlorinating PCBs. There is also a need for screening soil samples and the like to determine whether dioxygenase-susceptible PCBs are present, whereupon it might be of value to subject the soils to aerobic microbial treatment to degrade such PCBs.